Summary

The amyloid precursor protein (APP) is a type I transmembrane protein of
unknown physiological function. Its soluble secreted form (sAPP) shows
similarities with growth factors and increases the in vitro proliferation of
embryonic neural stem cells. As neurogenesis is an ongoing process in the
adult mammalian brain, we have investigated a role for sAPP in adult
neurogenesis. We show that the subventricular zone (SVZ) of the lateral
ventricle, the largest neurogenic area of the adult brain, is a major sAPP
binding site and that binding occurs on progenitor cells expressing the EGF
receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In
vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies
antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase
the number of EGF-responsive progenitors through their increased
proliferation. Conversely, blocking sAPP secretion or downregulating APP
synthesis decreases the proliferation of EGF-responsive cells, which leads to
a reduction of the pool of progenitors. These results reveal a new function
for sAPP as a regulator of SVZ progenitor proliferation in the adult central
nervous system.

Introduction

Aβ peptides found in senile plaques accompanying Alzheimer's disease
(AD) are formed by the amyloidogenic processing of a type-one integral
membrane protein, the amyloid precursor protein (APP)
(De Strooper and Annaert, 2000;
Small et al., 2001). This
processing requires cleavage at two distinct sites by the β and γ
secretases. Considerable effort has been devoted to understanding the
amyloidogenic processing of APP in relation to neurodegeneration and dementia,
but the physiological functions of APP remain unclear. The ubiquitous
expression of APP during development and in several adult tissues, along with
its high degree of conservation across phyla, suggests a fundamental role in
cellular physiology. In the central nervous system, the increase of APP
expression during development coincides with the peak of neuronal
differentiation and neurite outgrowth
(Hung et al., 1992;
Salbaum and Ruddle, 1994).
APP-null mutant mice show diverse non-lethal defects, including reduced brain
weight and reduced size of forebrain commissures
(Zheng et al., 1995;
Li et al., 1996;
Magara et al., 1999). However,
a clear interpretation of these mutant phenotypes has been hampered by a
possible functional compensation by other APP family members, the amyloid
precursor like proteins, APLP1 and APLP2
(Slunt et al., 1994;
Wasco et al., 1992).
Accordingly, combined APP/APLP2 invalidation leads to perinatal death
(Heber et al., 2000;
von Koch et al., 1997).

In addition to its still elusive role as a membrane-bound protein, APP can
be cleaved to produce soluble derivatives with biological activity. In
non-pathological situations, APP is predominantly cleaved by theα
-secretase within the β-amyloid sequence, to release a soluble
form of APP, sAPP (see Fig.
3A). sAPP is normally present in brain tissues and circulates in
the cerebrospinal fluid (CSF) (Palmert et
al., 1989). A number of in vitro studies have attempted to uncover
sAPP neuronal functions, showing that sAPP enhances synaptogenesis, neurite
outgrowth, cell survival and cell adhesion
(Mattson, 1997). These
growth-promoting properties, together with its structural similarities with
cysteine-rich growth factors (Rossjohn et
al., 1999), suggest that sAPP may function as a growth factor in
vivo. Interestingly, infusion of sAPP into the brain increases synaptic
density and improves memory retention
(Meziane et al., 1998;
Roch et al., 1994) and levels
of sAPP are decreased in the CSF of individuals with AD
(Lannfelt et al., 1995;
Van Nostrand et al., 1992).
Moreover, sAPP stimulates proliferation of neural stem cells isolated from the
embryonic brain (Hayashi et al.,
1994; Ohsawa et al.,
1999).

Changes in sAPP concentration modify the number of SVZ progenitors through
regulation of their proliferation. (A-D) Mice were intraventricularly infused
with human IgG (control), sAPP-Fc (sAPP), 0.09% NaCl (saline), batimastat
(bat), batimastat and sAPP (bat+sAPP), sense or antisense APP oligonucleotides
(S, AS). In A and C, NS were prepared from the SVZ ipsilateral to the side of
infusion and counted. In B and D, BrdU was injected 1 hour before sacrifice
and the percentage of BrdU-positive EGFR immunoreactive cells on the side of
infusion was counted. Results are shown as mean±s.e.m. of six infused
mice. (A,B) Augmentation of sAPP concentration leads to an increased number of
EGF-responsive NS progenitors in the SVZ (A) and to their increased
proliferation (B). (C) Reduction of sAPP secretion by batimastat or of APP
synthesis by antisense oligonucleotides decreases the number of EGF-responsive
NS progenitors. (Bottom panel) APP immunoblotting of the SVZ from two sense
(S) or two antisense (AS) oligonucleotide-infused mice reveals a 30% reduction
in APP levels in AS. (D) Reduction of APP synthesis through antisense
oligonucleotide infusion downregulates EGFR immunoreactive cell proliferation
and sAPP compensates this downregulation. (E) Ara-C infusion was followed by a
3 day intraventricular infusion of control IgG (Ara-C and IgG) or 6 E10
antibody (Ara-C and 6 E10) and the total number of BrdU-positive cells was
counted in whole-mount preparations of the anterior SVZ. During SVZ
regeneration, reduction of circulating sAPP downregulates EGF-responsive cell
proliferation.

The existence of adult neural stem cells and progenitors raises the
possibility that sAPP may also have an effect on neurogenesis in the adult.
The SVZ extends along the length of the lateral ventricle and, in this region,
neural stem cells retain the capacity to produce new neurons throughout
adulthood (Alvarez-Buylla and
Garcia-Verdugo, 2002). In rodents, newly produced neuroblasts
migrate rostrally toward the olfactory bulb where they differentiate into
granular and periglomerular interneurons. Studies of SVZ cells and of their
lineage relationships have shown that SVZ astrocytes (type B cells) give rise
to rapidly dividing transit amplifying precursors (type C cells) which in turn
give rise to migrating neuroblasts (type A cells)
(Doetsch et al., 1999a).
Members of the epidermal growth factor (EGF) family stimulate the
proliferation of a subpopulation of cells in the SVZ in vivo
(Craig et al., 1996;
Doetsch et al., 2002;
Kuhn et al., 1997;
Tropepe et al., 1997). These
EGF-responsive cells have recently been identified as the transit amplifying C
cells (Doetsch et al., 2002).
They can be grown in culture as neurospheres (NS)
(Doetsch et al., 2002), which
exhibit stem cell properties: they self-renew in the presence of EGF and
differentiate into neurons and glia upon EGF removal
(Morshead et al., 1994;
Reynolds and Weiss, 1992).

We show that the SVZ is a major binding site for sAPP in the adult brain
and that sAPP binds EGF-responsive cells. In vitro, EGF stimulates sAPP
secretion by NS cells and sAPP is required for full EGF mitogenic activity. In
vivo, modulating levels of sAPP results in modifications of the number of
EGF-responsive cells composing the SVZ transit amplifying compartment through
regulation of their proliferation. This work therefore provides strong
evidence for a function of sAPP in adult neurogenesis.

Materials and methods

sAPP-Fc production

Plasmids encoding human sAPP (695 amino acid form), sAPLP1 (up to amino
acid 584) or sAPLP2 (up to amino acid 680 of APLP2 571) fused to the Fc
fragment of human IgG1 were transfected into Cos cells. Purification of
sAPP-Fc (or sAPLP1/2-Fc) from the medium was performed on a protein
A-sepharose column by standard procedures.

For whole-mount staining, the mice were perfused with 2% paraformaldehyde,
the lateral wall of the lateral ventricle (injected side) was dissected and
immunohistochemistry was performed as described
(Doetsch et al., 1999b).

Infusions in vivo

sAPP-Fc (200 μg/ml), sAPLP2-Fc (200 μg/ml), human IgG (200 μg/ml,
Sigma), batimastat (100 μM, Glaxo Smith Kline), APP oligonucleotides [20μ
M, designed –10 to +10 around the mRNA initiation site and coupled
to penetratin (Derossi et al.,
1998)] or APLP2 oligonucleotides (20 μM, designed –3 to
+15 around the mRNA initiation site and coupled to penetratin) were diluted in
0.09% NaCl. At least six adult mice for each condition were infused
continuously for 3 days into the lateral ventricle (coordinates:
anterior/posterior, 0; lateral, 0.8 mm; dorsal, 2 mm, relative to bregma)
using micro-osmotic pumps (1 μl/hour, model 1003, Alzet). It is assumed
that the solution in the pump is diluted at least ten times upon infusion in
the CSF.

Quantification of proliferation

For quantification of EGF-responsive cell proliferation, infused animals
(n=3 for each condition) were injected intraperitoneally with BrdU (1
mg in 100 μl) 1 hour before perfusion with 4% paraformaldehyde. Brains were
removed, postfixed for 2 hours and cryoprotected in 20% saccharose overnight.
They were frozen and 10 μm thick sections were cut using a cryostat.
Frontal sections (20 per brain) at different levels of the SVZ (between +1 mm
and 0mm anterior to bregma) were double-labeled for EGFR (sheep anti-human
EGFR, 1:100, Upstate Biotechnology) and BrdU (rat anti-BrdU, Accurate
chemical, 1:200), and mounted with DAPI-containing mounting medium. Clearly
identifiable EGFR immunopositive cells were scored individually for their BrdU
immunoreactivity under the 100× objective of a Leica fluorescence
microscope.

For quantification of BrdU-positive cells in whole-mount preparations,
cells were counted on a predefined area corresponding to the anterior
subventricular zone using a computerized Biocom analysis system.

Neurosphere culture and assays

NS were prepared, cultured in serum-free medium with EGF (20 ng/ml),
passaged and differentiated to assess their multipotentiality as described
(Doetsch et al., 1999a). All
experiments were performed on NS that had been passaged several times.

To stain sAPP-binding sites on NS cells, 8-day-old in vitro (DIV) NS were
plated onto poly-L-ornithin (15 μg/ml) and laminin (10 μg/ml) coated
chamber-slides (16 well Lab-Tek, Nunc) in medium without EGF for 4 hours.
Cells were fixed in 4% paraformaldehyde for 15 minutes, rinsed in PBS and
blocked in PBS with 10% fetal calf serum for 1 hour. They were incubated
overnight at 4°C in sAPP-Fc diluted to 10 μg/ml in PBS with 10% fetal
calf serum. The signal was developed as for sections. Cells were mounted with
DAPI-containing vectashield mounting medium (Vector).

To measure sAPP secretion by NS, 8 DIV NS were centrifuged, resuspended in
fresh medium and plated into 96-well plates (approximately 1000 NS per well in
50 μl medium). After 15 hours, NS were centrifuged and the medium and
cellular pellet from each separate well were processed independently for
immunoblotting using the 22C11 antibody (Chemicon). The signal was developed
using the application Image Gauge.

Results

sAPP binding sites are present in the SVZ

APP is ubiquitously expressed in the brain and immunohistochemical staining
of APP on brain sections shows that SVZ cells also express APP
(Fig. 1A). Detection of sAPP
signal transduction in different cell types
(Greenberg et al., 1994;
Pietrzik et al., 1998)
supposes the existence of a membrane receptor for sAPP. Saturable sAPP-binding
sites have been detected in several cell lines
(Hoffmann et al., 1999) but no
receptor has yet been identified. We used a fusion protein consisting of the
Fc domain of human immunoglobulin G (IgG) linked to sAPP (sAPP-Fc) to detect
sAPP-binding sites on brain tissue sections. Interestingly, sAPP-Fc, but not
control human IgGs, prominently stained the surface of SVZ cells
(Fig. 1B). Double-labeling
showed that SVZ EGFR immunoreactive cells (type C cells) express sAPP-binding
sites (Fig. 1C-E). sAPP-binding
sites are also present on SVZ neuroblasts (type A cells), identified by
PSA-NCAM staining (Fig. 1F-H).
S100 immunopositive astrocytes (type B cells) and ependymal cells do not
harbor sAPP binding sites (Fig.
1I-K). Under the staining conditions used, we did not detect
sAPP-binding sites in other regions of the brain. Notably, we were unable to
detect sAPP-binding in the hippocampus, the second major area where adult
neurogenesis occurs (Kempermann,
2002).

To verify if the prominent SVZ staining by sAPP-Fc and the absence of
staining in other brain regions, in particular the hippocampus, reflects a
heterogeneous penetration of sAPP-Fc within brain tissues, we used another
Fc-tagged protein, L1-Fc (Doherty et al.,
1995). In the same binding assay, L1-Fc binds sites in diverse
regions of the brain, including the SVZ, the hippocampus and the cortex
(Fig. 1L-N). This confirms that
the detection of sAPP-binding sites in the SVZ is specific and that SVZ cells
express high amounts of sAPP-binding sites. The possibility cannot be
excluded, however, that low levels of binding sites or binding sites with
lower affinities and below the limit of detection of our method are expressed
in other brain regions.

sAPP participates in the EGF-induced proliferation of adult SVZ
progenitors in vitro

The two likely cellular targets of infused sAPP are neuroblasts (Type A
cells) and EGF-responsive cells (Type C cells), which both express
sAPP-binding sites. We have focused the present study on the effect of sAPP on
EGF-responsive cells. These cells can be cultured and, in the presence of EGF,
they proliferate and form small aggregates called neurospheres (NS). We first
verified if NS cells can bind sAPP. Detection of sAPP-Fc on plated NS cells
showed that they do bind sAPP, with a punctuate staining pattern at the cell
surface (Fig. 2B). To determine
whether NS cells also secrete sAPP, we immunoblotted media conditioned by NS
cultured with 0, 2 or 20 ng/ml of EGF. EGF causes a concentration-dependent
increase in the release of sAPP (Fig.
2C, upper panel). By contrast, cellular APP remains approximately
constant at the three EGF concentrations
(Fig. 2C, lower panel). It is
noteworthy that a similar growth factor-induced sAPP release has been shown in
different cell lines (Schubert et al.,
1989; Slack et al.,
1997).

sAPP participates in the EGF-induced proliferation of neurospheres. (A)
Schematic structure of human APP (695 amino acid form) indicating the
positions of the epitopes of 22C11 and 6 E10 antibodies. α-sAPP, soluble
APP; Aβ, amyloid β-peptide; TM, transmembrane region. (B)
sAPP-binding sites (red) are detected on plated undifferentiated NS as a
punctuate membrane staining. Nuclei are counterstained with DAPI. Scale bar: 5μ
m. (C) A constant number of NS were seeded in media with different
concentrations of EGF (0, 2 or 20 ng/ml). Secreted (upper panel) and cellular
(lower panel) APP were detected by immunoblotting. Secretion of sAPP is
augmented by increasing concentrations of EGF (2 ng/ml, 3.5 fold; 20 ng/ml,
5.9 fold increase relative to EGF-free medium) while cellular APP remains
largely constant. (D) Percentage of BrdU-positive NS cells after 15 hours of
culture in medium containing EGF (2 ng/ml) and BrdU without (control) or with
antibodies directed against sAPP (22C11, 6E10). Both antibodies decrease
proliferation of NS cells. This decrease is abolished by pre-adsorption of the
antibodies with their epitopes or by co-incubating 22C11 and sAPP. Results are
shown as mean±s.e.m. of three independent experiments.

This observation suggested that sAPP might participate in the EGF-induced
proliferation of NS cells. To verify this point, NS were cultured for 15 hours
with bromodeoxyuridine (BrdU), a marker of DNA synthesis (phase S), in the
presence of the 22C11 antibody that binds a small domain between amino acids
66-82 in the APP N terminus (Fig.
2A). The addition of 22C11 antibody leads to a significant
decrease in the percentage of BrdU-positive cells (42±4%) when compared
with control conditions without antibody (58±4%) or to conditions in
which the antibody was pre-adsorbed onto its epitope (57±4%). This
decrease in proliferation was also reversed when sAPP (10 μg/ml) was added
to the medium with the antibody (56±3%)
(Fig. 3D, t-test
P<0.001).

The 22C11 antibody has been shown to induce intracellular signaling through
binding to membrane APP, thus mimicking the action of an unknown endogenous
ligand (Brouillet et al., 1999;
Okamoto et al., 1995). We
therefore followed the proliferation of NS cells in the presence of a
different antibody (6 E10) that binds another region comprised between amino
acids 597 and 613 of the APP N terminus
(Fig. 2A). Addition of this
antibody also leads to a decrease in the percentage of BrdU-positive cells
(29±3%) and this decrease is also prevented by a pre-incubation of the
antibody with its epitope (57±2%)
(Fig. 2D, t-test
P<0.001). The similar decrease of proliferation provoked by two
antibodies recognizing two distinct epitopes suggests that they act by
sequestering the sAPP released into the medium upon EGF addition and not
through a ligand-like signaling activity. We also verified that the decrease
in BrdU incorporation does not reflect a cell survival effect by counting the
number of apoptotic TUNEL-positive cells. We found that the percentage of
apoptotic cells remained unchanged after the addition of either anti-APP
antibody (control, 7.2±1.1%; 22C11, 7.4±1.8%; 6 E10,
7.8±1.5%).

The effect of sAPP on NS cell differentiation was tested by adding sAPP-Fc
or anti-APP antibodies in conditions that induce the differentiation of NS
cells into neurons, astrocytes and oligodendrocytes. Compared with control
conditions, the relative percentages of each differentiated cell types were
found unaltered by these treatments (not shown), suggesting that sAPP does not
play a role in NS differentiation in vitro.

Taken together, these results suggest that the EGF-induced proliferation of
NS cells is partly dependent on the EGF-induced release of sAPP into the
medium. However, sAPP is necessary but not sufficient as it does not induce
cell division in EGF-free medium. Finally, the addition of exogenous sAPP-Fc
(0.1 to 20 μg/ml) to EGF-containing medium did not lead to a further
increase in NS cells proliferation (not shown), a finding that may be
explained by a saturating effect of the EGF-induced secretion of endogenous
APP. We thus decided to turn to the in vivo situation to test the effects of
sAPP gain and loss of function on the proliferation of adult progenitors.

To determine whether sAPP also acts on EGF-responsive SVZ cells in vivo, we
unilaterally infused sAPP-Fc (200 μg/ml) or human IgG (same concentration)
into the lateral ventricle for 3 days and prepared NS from the SVZ ipsilateral
to the side of the infusion. The number of NS growing from each SVZ reflects
the total number of NS precursors present in the SVZ at the time of dissection
(Morshead et al., 1994). The
average number of NS obtained from the SVZ of control mice infused with human
IgG was 1940±89. This number, and therefore the number of progenitors
present in vivo, increased up to 2444±49 after infusion of sAPP-Fc
(Fig. 3A, t-test
P<0.01).

These NS were tested for their ability of self-renewal and their
differentiation properties and showed no difference as compared to control NS
(not shown). Because NS precursors are mainly EGF-responsive Type C cells
(Doetsch et al., 2002), this
suggested an increase in the number of these progenitors. To assess whether
this increase reflected their enhanced proliferation, BrdU was injected, 1
hour before sacrifice, into animals that had been infused for 3 days and the
cells double-stained for BrdU and EGFR were counted. A larger fraction of
EGF-responsive cells were BrdU-positive in sAPP-Fc infused mice
(64.9±6%) compared with control mice infused with human IgG
(43±2%) (Fig. 3B,
t-test P<0.01). This suggests that sAPP positively
regulates the proliferation of EGF-responsive SVZ adult progenitors in
vivo.

The α-secretase activity, which allows the production of sAPP from
APP, can be inhibited by hydroxamic acid-based zinc metalloprotease inhibitors
such as batimastat (Parvathy et al.,
1998).

Therefore, to evaluate the effect of a reduction of sAPP concentration on
EGF-responsive SVZ cells, we unilaterally infused batimastat (100 μM) into
the lateral ventricle for 3 days and prepared NS from the SVZ, as above. The
number of NS was reduced from 1856±36 in control mice to 626±34
after batimastat infusion (Fig.
3C, t-test P<0.001). Because batimastat
probably inhibits the cleavage of several membrane proteins in addition to
APP, we tested the specificity of the effect by co-infusing sAPP-Fc (200μ
g/ml) and batimastat before preparing NS. As shown in
Fig. 3C, sAPP addition fully
reversed the effect of batimastat, restoring the number of NS back to control
levels (1948±60). This strongly suggests that the reduction of SVZ
progenitors upon batimastat infusion is a consequence of a decrease in the
cleavage of APP, sAPP and not of another unknown factor.

As an independent means of assessing the effect of sAPP reduction, we
infused an antisense oligonucleotide (20 μM) directed against APP and
linked to penetratin for internalization
(Derossi et al., 1998). This
linkage catalyses an extremely efficient addressing of the oligonucleotide
into the cell cytoplasm and nucleus
(Allinquant et al., 1995). It
allows the use of low concentrations of oligonucleotides, thus preventing
undesirable effects of the polyanionic nature of oligonucleotides.
Quantification of APP in the SVZ by immunoblot shows a 30% reduction in
antisense- as compared to sense-infused animals
(Fig. 3C). The number of NS
growing after infusion of sense oligonucleotides was 2167±18, whereas
infusion of antisense oligonucleotides led to a reduction to 1229±34
(Fig. 3C, t-test
P<0.01). This indicates that a reduced synthesis of APP leads to a
reduction in the number of EGF-responsive progenitors.

To evaluate if, as anticipated from the in vitro studies, this reduction of
the pool of progenitors was a consequence of their reduced proliferation, we
injected BrdU into infused animals and calculated the percentage of
EGF-responsive cells labeled with BrdU, as above. A lesser fraction of
EGF-responsive cells were BrdU-positive in antisense-infused
(33.8±1.4%) compared with sense-infused mice (42.7±1.8%)
(Fig. 3D, t-test
P<0.01). Because antisense treatment reduces APP synthesis and
thus both the transmembrane form and its released extracellular domain (sAPP),
we tested whether sAPP could rescue this reduction of proliferation. This was
indeed the case as the percentage of BrdU-positive EGFR immunoreactive cells
was above control levels in animals co-infused with antisense and sAPP
(54±2.2%) (Fig. 3D,
t-test P<0.01). This suggests that the reduction of
proliferation of SVZ progenitors upon antisense infusion results from a
decrease in sAPP and confirms that a reduction of sAPP level leads to reduced
proliferation of adult SVZ progenitors.

Taken together, these data strongly suggest that sAPP regulates the
proliferation of SVZ progenitors. However, as illustrated in
Fig. 1, sAPP-binding sites are
present in vivo both on Type C cells (EGFR-expressing progenitors) and Type A
cells (PSA-NCAM-expressing neuroblasts). To verify that sAPP directly acts on
type C cells and that sAPP-induced progenitor proliferation does not happen
through an indirect action on type A cells, we took advantage of the
well-defined system of SVZ regeneration after infusion of the antimitotic drug
cytosine-β-D-arabinofuranoside (Ara-C)
(Doetsch et al., 1999b). In
this model, after elimination of all Type C and A cells by Ara-C, the SVZ
regenerates rapidly with a sequential reappearance of type C (2 days after
termination of Ara-C treatment) and A cells (4 days after treatment). This
system allows one to follow the response of type C cells in the absence of
neuroblasts. We thus infused antibodies directed against sAPP (6 E10, 200μ
g/ml) or control IgG (same concentrations) during the first 3 days of
regeneration before the reappearance of type A cells. BrdU was injected 1 hour
before sacrifice to count the total number of proliferating cells on
whole-mount preparations of the anterior SVZ. The number of BrdU-positive
cells was significantly reduced in antibody-infused animals (557±78)
when compared with control animals (1235±45)
(Fig. 3E, t-test
P<0.01). This strongly suggests that a reduction of circulating
sAPP downregulates type C cell proliferation and that this reduction is a
direct effect of sAPP on type C cells.

sAPLP2 is functionally redundant with sAPP in the SVZ

This study points towards a role for sAPP in regulating the proliferation
of cells composing the SVZ transit amplifying compartment. APP is a member of
a larger gene family, including the two amyloid precursor-like proteins APLP1
and APLP2 (Slunt et al., 1994;
Wasco et al., 1992). APLPs are
highly homologous to APP and can be processed in a similar way, leading to the
secretion of their ectodomains (sAPLP)
(Paliga et al., 1997;
Slunt et al., 1994). It is
thus possible that sAPLPs and sAPP have redundant functions in the SVZ.

As illustrated in the western blot of
Fig. 4C, both APLP1 and APLP2
are expressed in the SVZ. However, when Fc-tagged sAPLPs were used to detect
if binding sites are present on brain tissue sections, it was found that
sAPLP2-Fc stained SVZ cells similarly to sAPP-Fc
(Fig. 4B), whereas sAPLP1-Fc
showed no detectable binding (Fig.
4A). We therefore assessed whether sAPLP2 could stimulate the
proliferation of EGF-responsive cells by infusing sAPLP2-Fc in the lateral
ventricle and counting the number of NS growing from the infused SVZ. The
average number of NS obtained from control mice infused with human IgG was
1862±58 and increased up to 3050±50 upon sAPLP2-Fc infusion
(Fig. 4D, t-test
P<0.01). Conversely, infusion of a penetratin-linked antisense
oligonucleotide directed to APLP2, which leads to a 30% decrease in APLP2
synthesis (Fig. 4D), was
paralleled by a significant decrease in the number of NS (1276±104).
This decrease was not observed with a control sense oligonucleotide
(1972±68) (Fig. 4D,
t-test P<0.01).

Increasing or decreasing sAPLP2 concentrations thus leads, respectively, to
an increase or a decrease in the number of SVZ EGF-responsive progenitors, a
result very similar to that obtained with sAPP. This suggests that both
molecules serve similar and possibly redundant physiological functions in the
SVZ.

Discussion

This study points towards a new site of action and physiological function
of sAPP in the adult mammalian brain. We show that sAPP-binding sites are
prominently present in the SVZ on Type C cells characterized by the expression
of the EGFR and on type A cells stained with the anti-PSA-NCAM antibody. A
role for sAPP in the control of type C cell proliferation is supported by in
vitro and in vivo experiments. In vitro, NS proliferation is dependent on the
presence of EGF in the culture medium but also on that of sAPP released from
the cells upon EGF addition. This indicates that sAPP plays a part in adult
EGF-responsive cell proliferation and is further supported by in vivo
approaches. Indeed, in vivo gain and loss of function of sAPP increases and
decreases, respectively, the number of EGF-responsive SVZ progenitors
composing the transit amplifying compartment (C cells) through modulation of
their proliferation. It can thus be proposed that sAPP acts as a paracrine or
autocrine factor mediating the proliferative effect of EGF on EGF-responsive
cells. As a consequence, changes in the levels of circulating sAPP are likely
to have direct consequences on the SVZ neurogenic potential by modifying the
number of cells composing its transit amplifying compartment.

Specificity of sAPP binding and activity

The binding of sAPP-Fc on both Type A and Type C cells clearly indicates
that the protein probably has multiple sites of action and may not only
influence the rate of division of Type C cells but also the division and
migration of Type A cells. Although we do not preclude an effect on Type A
cells, the experiments on Ara-C-treated animals demonstrate that the effect on
Type C cell proliferation is not an indirect consequence of an effect on type
A cells. This justifies the focus put, in this study, on the role of sAPP in
Type C cell growth regulation.

Another aspect of specificity is the lack of binding of sAPP-Fc in the
dentate gyrus, a second site of neurogenesis in the adult CNS. The absence of
sAPP-Fc binding is not due to a lack of penetration of sAPP-Fc because a
similar reagent, L1-Fc, binds both SVZ and hippocampal cells. This means that,
at least in the adult, the number of sites or their affinity is lower than in
the SVZ. This correlates with an absence of biological activity of sAPP-Fc, as
monitored by the number of BrdU-labeled cells in the dentate gyrus after
infusion of sAPP-Fc [on 10 representative sections, this number was
51±5 in sAPP-Fc infused animals and 54±3 in control animals
(mean±s.e.m., n=3)]. This finding is apparently at odds with
some in vitro studies on hippocampal cells showing that sAPP or smaller
peptides derived from sAPP have a trophic action or influence neuronal
excitability (Mattson, 1997).
Some of these studies provide evidence for an underlying signal transduction
pathway suggesting the existence of a receptor for sAPP. However, most of the
above-mentioned studies have been performed on embryonic cells or on cell
lines and not on adult brain or NS cells derived from adult brain.

The absence of sAPP-binding sites in the dentate gyrus of the hippocampus
and its ineffectiveness at promoting proliferation in this region also
suggests that, in the adult, its role in neurogenesis could be limited to the
SVZ. This is consistent with the fact that dentate gyrus neurogenesis is
regulated differently from SVZ neurogenesis. In particular, the dentate gyrus
progenitors are more restricted in their potentiality than SVZ progenitors and
cannot be cultured as NS (Seaberg and van
der Kooy, 2002). In line with this, an EGF-responsive transit
amplifying compartment similar to the SVZ type C cells, shown here to be
regulated by sAPP, is missing in the dentate gyrus
(Seri et al., 2001).

A function for sAPP in regulating the proliferation of different cell types
has been proposed in previous studies performed on cell lines
(Hayashi et al., 1994;
Hoffmann et al., 2000;
Ohsawa et al., 1999;
Pietrzik et al., 1998;
Saitoh et al., 1989).
Interestingly, sAPP has been described as an autocrine regulator of epidermal
basal cell proliferation (Hoffmann et al.,
2000), which, similar to SVZ progenitor proliferation, is under
the control of TGFα, a member of the EGF family that acts though the EGF
receptor. Cultured keratinocytes also exhibit sAPP-binding sites similar to
the ones we observed in the SVZ or on cultured NS. In line with the results
presented here, sAPP was described to stimulate proliferation of embryonic
neural stem cells (Hayashi et al.,
1994; Ohsawa et al.,
1999). Accordingly, we could detect sAPP-binding sites not only on
adult NS cells and SVZ but also on NS cells cultured from the embryonic brain
(Fig. 5). Consequently, whereas
this study is limited to adult neurogenesis, it is possible that sAPP also
triggers progenitor proliferation at early developmental stages. This
possibility is supported by the reduced brain volume of mice deleted for APP
(Zheng et al., 1995) and by a
study on transgenic mice overexpressing a mutant form of APP
(Bondolfi et al., 2002),
revealing that these mice have 10-15% more cortical neurons than wild-type
control mice. This, in view of the present results, could be a consequence of
an increased neurogenesis consecutive to an increase in sAPP
concentration.

sAPP and transmembrane APP

The in vitro experiments demonstrate that the proliferation of NS in
response to EGF is blocked in the presence of two antibodies that recognize
two distinct epitopes in the N-terminal domain of APP. The antibodies could
either act as agonists for transmembrane APP, as described for 22C11
(Brouillet et al., 1999;
Okamoto et al., 1995), or by
sequestering sAPP and thus preventing its availability to receptors. The
latter hypothesis is more likely because the two antibodies recognizing two
different epitopes have similar effects and it has never been reported that
6E10 mimics an APP ligand.

Also in favor of a direct role for sAPP as opposed to transmembrane APP, is
the effect of inhibition of sAPP formation through batimastat infusion, which
is fully reversed by the infusion of sAPP. In addition, antisense infusions
are extremely demonstrative as the decrease in transmembrane APP is
compensated by sAPP in the absence of a restoration of the normal
concentrations of the transmembrane form. These data, together with the
specificity of action of sAPP on the SVZ, which presents the highest
concentration of sAPP-binding sites, support the idea that it is sAPP that
triggers Type C cell proliferation and that, as far as this activity is
concerned, transmembrane APP acts as a precursor. This, indeed, does not
preclude other functions associated with this transmembrane form.

sAPP and sAPLP2 as EGF/TGFα co-factors

The signaling pathway triggered by sAPP was not investigated here. It is
important to note that, in the model that we propose, the activity of EGF
requires that sAPP be present in the medium. sAPP is necessary, but not
sufficient for EGF activity (it cannot replace EGF) and thus, in this model,
acts as an EGF co-factor for Type C cell proliferation. It would be of great
interest, in the future, to identify which pathways are co-regulated by the
two molecules. To this extent, it is interesting that two studies have
reported a stimulation of MAP kinase activity by sAPP
(Greenberg et al., 1994;
Pietrzik et al., 1998). It is
also striking that, in vitro, the addition of sAPP in the presence of EGF does
not further enhance NS proliferation. This situation differs from that
observed in vivo, where the infusion of sAPP has a growth-promoting effect.
This difference between the in vitro and in vivo situations might be due to
the low levels of sAPP at the vicinity of their target cells, possibly owing
to its fast clearance in the CSF. If so, sAPP would be a limiting factor for
Type C cell proliferation in the adult SVZ.

We show that sAPLP2 also binds SVZ cells and acts similarly to sAPP on
EGF-responsive progenitors, demonstrating a physiological function for sAPLP2.
These results confirm the functional redundancy between APP and APLP2,
postulated on the basis on their high degree of homology (up to 72% identical
in the cysteine-rich regions of their ectodomains)
(Wasco et al., 1992) and from
the lethal phenotype of the double knockout mice
(Heber et al., 2000;
von Koch et al., 1997) as
opposed to minor phenotypes of viable single knockouts
(Li et al., 1996;
Zheng et al., 1995). This
redundancy associated with the structural similarities between the two
proteins and the fact that sAPP is a limiting factor, as proposed above,
provides a rational for the fact that the two proteins have the same effects
on Type-C cell proliferation and suggests that they act on the same receptors.
The absence of compensation between the two molecules in the adult
loss-of-function experiments is also in agreement with their limiting
concentrations. This is not contradictory with the compensation observed in
the genetic deletions as gene deletion in the embryo sets up permanent
compensation phenomena not observed in the acute adult treatments used in this
study.

sAPP and AD

The present findings might have implications for the understanding of AD.
The extent and significance of adult neurogenesis in the human brain is a
matter of controversy. However, human and rodent SVZ cells express similar
markers, including the EGFR (Weickert et
al., 2000). A subset of human SVZ cells have been shown to divide
(Eriksson et al., 1998) and
the human SVZ is neurogenic in vitro
(Kirschenbaum et al., 1994).
This supports the view that SVZ neurogenesis is similarly regulated in all
mammals. In the light of the reduction in sAPP concentrations in brains from
individuals with AD (Lannfelt et al.,
1995; Van Nostrand et al.,
1992), the present data that link sAPP levels and the number of
SVZ progenitors, suggest that a deficit in neurogenesis could play a role in
some aspects of AD pathogenesis. Interestingly, individuals with AD show very
early odor discrimination defects (Nordin
and Murphy, 1998) as do mutant mice with reduced SVZ neurogenesis
(Gheusi et al., 2000). Even
though impaired olfaction in AD could be a consequence of cortical
neurodegeneration, reduced olfactory bulb neurogenesis could participate in
this phenomenon. Similarly, it is interesting that the primate SVZ gives rise
to neurons destined to the amygdala and pyriform cortex
(Bernier et al., 2002), two
regions important for emotions and memory and atrophied in AD
(Callen et al., 2001). Added to
the implication of adult neurogenesis in learning and memory
(Scharff, 2000), this
observation suggests that some cognitive impairments in AD might result from a
defect in SVZ neurogenesis.

Acknowledgments

We thank M. Volovitch for help with plasmid constructs, E. Brouillet for
construction of the sAPP-Fc vector, G. Rougon for antibodies to PSA-NCAM,
Glaxo Smith Kline for Batimastat and Patrick Doherty for the L1-Fc construct.
This work was supported by CNRS and ENS; and by grants from the Association
pour la Recherche sur le Cancer (ARC-5168), from the GIS-Infections à
Prions/A32 and from Aventis Pharma industry.

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